Present evidence would suggest that an important control mechanism in the hepatic microsomal ethylmorphine N-demethylase is the activation of the NADPH-cytochrome P-450 reductase by ethylmorphine. When NADPH is the sole source of reducing equivalents, this process appears to regulate the rate at which electrons are transported down the electron chain to provide the first reducing equivalent necessary for the reduction of the oxygen which participates in the hydroxylation and subsequent N-demethylation of the ethylmorphine. It is unclear at this time as to 1) whether this site binds to the hydrophonic or hydrophillic substituents of the drug and 2) whether this process is important for other hydroxylases. I propose to investigate the first question by studying the kinetics of the activation of the reductase for a series of morphine analogues in which alkyl groups of increasing chain length are substituted onto the nitrogen and the 3-hydroxyl. The second question shall be examined by determining the stoichiometry for these same substrates between the activation and the two dealkylations (N- and O-dealkylation) which preliminary evidence would suggest are catalyzed by separate classes of hydroxylase. Similarly, the formation of the diastereoisomeric pairs from the enantiomers of pentobarbital will be compared to the kinetics of activation by each of the enantiomers of the reductase. The effect of inhibitors which appear to characterize these classes of hydroxylases will be examined for their effect on the ratio of products and on the kinetics of the activation. Solubilized and purified microsomal enzymes will be examined for their ability to metabolize the various substrates to determine the role of different enzymes in determining the rate and nature of metabolism of drugs. From these studies we hope to develop a clear model of the hepatic, microsomal mixed-function oxidases, and, in particular, to elucidate the role of the activation of the NADPH-cytochrome P-450 reductase in other hydroxylases.